Theophylline is a drug frequently administered for treatment of asthma and pulmonary diseases. For the drug to be used successfully without serious side-effects, it must be frequently and carefully monitored in a patient because it has a relatively narrow therapeutic range of use, that is, 1-2 mg/dl.
Numerous techniques have been used to determine the amount of theophylline in human serum. Most of these techniques have serious drawbacks. For example, known spectrophotometric methods require large sample volumes, extensive pretreatment and suffer from interferences by similarly structured xanthines, such as caffeine and theobromine. Known gas chromatographic methods are more specific, but require derivitization and are time consuming.
Nonisotropic immunoassay techniques are most frequently used because they provide rapid results and are simple to use. Although satisfactory sensitivity has been generally obtained with immunoassay techniques, it has been found recently that they may produce hightly elevated results depending upon a patient's renal condition and the specificity of the antibody used in the assay. Moreover, immunoassays require the use of generally costly reagents which have limited stability.
High performance liquid chromatography techniques are also known. These techniques vary in specificity depending upon whether pretreatment of the test sample is carried out. Organic extraction steps are necessary to improve the accuracy and specificity of the assay. Many chromatography methods are susceptible to interferences from a number of substances including some common antibiotics. Other disadvantages include the need for expensive instrumentation and a specialized technical staff to perform the assays.
It is known that theophylline can be determined by measuring its inhibitory effect on alkaline phosphatase activity. However, when assaying human biological fluids in this manner, it is known that endogenous alkaline phosphatase can affect the assay and render inaccurate results on the high side. Endogenous alkaline phosphatase must then be destroyed or removed in some manner prior to the assay to avoid this problem.
In a literature article by B. Vinet and L. Zizian [Clin. Chem., 25:8, pp. 1370-1372 (1979)], an assay for theophylline in human serum is described in which the drug was extracted from the serum sample using chloroform/isopropanol to separate the theophylline from an unknown quantity of endogenous alkaline phosphatase prior to the actual determination of theophylline. The amount of theophylline was determined at pH 9.4 by measuring the amount of inhibition of bovine alkaline phosphatase activity which occured due to the presence of theophylline. This assay has several serious drawbacks, however. It is limited to solution assays. Further, it is slow and tedious due to the multiple extraction steps required to separate endogenous alkaline phosphatase from theophylline prior to actual determination of the drug.
A significant advance in the art is described in commonly assigned and copending U.S. Ser. No. 692,473, filed Jan. 18, 1985 by Norton and entitled Analytical Element and Method For Determination Of Theophylline By Enzyme Inhibition. The element described and claimed therein, however, has an isoenzyme for alkaline phosphatase and a buffer for maintaining the pH at 9 or less during the assay located in the registration layer. That layer is located adjacent to the support. There is no buffer described in the spreading layer of the element.
While this element provides a simple and rapid assay for theophylline in which endogenous alkaline phosphatase exhibits a reduced effect, there is a need for further improvement in that assay. The element described above has a limited rate range. Further, the element described above has reduced keeping properties which means that it is susceptible to enviromental conditions.